Cell Inhibition by HLA-Bw4 Ligand KIR3DL1 Polymorphisms That

KIR3DL1 Polymorphisms That Affect NK Cell Inhibition by HLA-Bw4 Ligand William Henry Carr, Marcelo Jorge Pando and Peter Parham This information is current as J Immunol 2005; 175:5222-5229; ; of September 25, 2021. doi: 10.4049/jimmunol.175.8.5222 http://www.jimmunol.org/content/175/8/5222 Downloaded from References This article cites 33 articles, 16 of which you can access for free at: http://www.jimmunol.org/content/175/8/5222.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

KIR3DL1 Polymorphisms That Affect NK Cell Inhibition by HLA-Bw4 Ligand1

William Henry Carr,2 Marcelo Jorge Pando, and Peter Parham2

The killer cell Ig-like receptor (KIR) gene family encodes MHC class I receptors expressed by NK cells and several T cell subpopulations. Factors contributing to human KIR haplotype diversity are differences in gene number, gene content, and allelic polymorphism. Whereas functional and clinical consequences of the ﬁrst two factors are established, knowledge of the effects of KIR gene polymorphism is limited to special cases in which signaling function is reversed or cell surface expression lost. In this study we use retrovirally transduced human cell lines to show that 3DL1*002 is a stronger inhibitory receptor for HLA-Bw4 ligands than 3DL1*007. Analysis of mutant 3DL1*002 and 3DL1*007 molecules demonstrates that residue 238 in the D2 domain and 320 in the transmembrane region contribute to the difference in receptor strength. Neither position 238 nor 320 is predicted

to interact directly with HLA-Bw4 ligand. This study also revealed that KIR3DL1 and LILRB1 both contribute to developing an Downloaded from inhibitory response to HLA-Bw4 ligands. The Journal of Immunology, 2005, 175: 5222–5229.

atural killer cell responses are regulated by arrays of these specificities have yet to be confirmed by biochemical or cel- inhibitory and activating receptors, several of which en- lular analysis. Although it is clear that KIR can distinguish be- N gage MHC class I or class I-like ligands. Some of the tween groups of allotypes of an HLA locus, a pressing and largely class I receptors are conserved and ubiquitously expressed, unanswered question is: do KIR allotypes differ in their recogni- http://www.jimmunol.org/ NKG2D for example, whereas others are variable and differen- tion of an HLA class I ligand? tially expressed (1, 2). Extreme examples of the latter category are In studying this question the KIR3DL1 locus has several exper- rodent Ly49 receptors and primate killer cell Ig-like receptors imental advantages such as high polymorphism (i.e., many distinct (KIR),3 functional analogues that have many genetic similarities, allotypes), defined HLA-B ligands, inhibitory function, and spe- but no structural homology (3). cific mAbs, which do not cross-react with other KIR. Moreover, The KIR locus and the leukocyte Ig-like receptor (LILR) locus flow cytometric analysis has shown that different KIR3DL1 alleles form part of the leukocyte receptor complex on human chromo- define different levels of cell surface expression, which points to some 19 (4). The KIR genes are closely packed and separated by the allotypes having functional difference (9). One special case is small, homologous intervening sequences. This organization facil- the 3DL1*004 allele, for which the protein is made but is retained by guest on September 25, 2021 itates unequal crossing over, one mechanism that has contributed within the cell (10); a second special case is KIR3DS1, which to the abundant variability in human KIR haplotypes. KIR haplo- segregates as an allele of the KIR3DL1 gene but has potential types differ in gene number (ϳ5–15) and content, importantly in activating rather than inhibitory function (11). So far KIR3DS1 has the relative numbers of genes encoding activating and inhibitory resisted biochemical and immunological characterization, but an receptors (5). Most of the KIR genes exhibit allelic polymorphism analysis of HIV-infected patients found the combination of and this serves to diversify KIR haplotypes further. KIR3DS1 and a presumptive Bw4 ligand to be correlated with KIR genes encode receptors that differ in specificity for HLA slower progression to AIDS (12). class I ligands and signaling potential. Well recognized are the The goal of the investigation we describe was to compare the specificities of KIR2DL1, 2DL2, 2DL3 and KIR2DS1 for HLA-C, functional recognition of HLA-Bw4 by two KIR3DL1 allotypes KIR3DL1 for HLA-B, and KIR3DL2 for HLA-A (6–8). Clinical that are both inhibitory receptors and expressed at the cell surface, correlations with infection, autoimmunity, and transplantation im- but which differ at selected positions. plicate other KIR as having common HLA class I ligands, but Materials and Methods Generation of KIRϩ NK leukemia cell lines and cultured NK Department of Structural Biology, Stanford University, Stanford, CA 94305 cells Received for publication April 8, 2005. Accepted for publication July 13, 2005. The costs of publication of this article were defrayed in part by the payment of page PBMC samples were obtained from healthy human donors by informed charges. This article must therefore be hereby marked advertisement in accordance consent under an Institutional Review Board approved protocol. 3DL1*007 with 18 U.S.C. Section 1734 solely to indicate this fact. (AF262973) was isolated as described (2) and subcloned into a retroviral 1 This work was supported by National Institutes of Health Grant AI22039 (to P.P.) vector pMX-puro (13), which is a gift from Dr. T. Kitamura (The Institute and by National Institutes of Health, National Institute of Allergy and Infectious of Medical Science, University of Tokyo, Tokyo, Japan). NK leukemia Diseases Grants K08 AI50779-01 and AI50779-02 (to W.H.C.). W.H.C. is a recipient (NKL) cells, a gift from Dr. M. Robertson (Department of Medicine, In- of nonduplicating support from the United Negro College Fund, Merck Science Ini- diana University, Indianapolis, IN) (14), were transduced to express KIR tiative Fellowship. using the Phoenix Helper dependent protocol as described by Gary ͗ ͘ 2 Address correspondence and reprint requests to Drs. Peter Parham and William Nolan ( www.stanford.edu/group/nolan/protocols/pro_helper_dep.html ). Henry Carr, Department of Structural Biology, Stanford University, Stanford, CA KIR3DL1 mutants were generated using the Quick Change site-directed 94305-5126. E-mail address: [email protected]. Department of Microbiology mutagenesis kit (Stratagene) according to the manufacturer’s instruc- and Immunology, University of California, San Francisco. E-mail address: tions. The NKL 3DL1*002 cell line, which was produced by transduc- [email protected] tion of NKL with the 3DL1*002 (U31416) pMX retroviral vector, was 3 Abbreviations used in this paper: KIR, killer cell Ig-like receptor; LILR, leukocyte generously provided by Dr. L. Lanier (University of California, San Ig-like receptor; NKL, NK leukemia; IOL, inhibition of lysis. Francisco, CA).

PE-labeled, NK cell receptor-specific Abs anti-KIR3DL1 mAb DX9 Results (DNAX); anti-KIR2DL1 mAb EB6 (Corixa); anti-CD94 mAb HP3D9 (BD To compare the function of KIR3DL1 allotypes we used NKL Biosciences); anti-NKG2A mAb Z199 (Corixa); and unconjugated anti- LILRB1 mAb HP-F1, a gift from M. Lo´pez-Botet (Universitat Pompeu cells transduced with natural and mutant forms of KIR3DL1. Al- Fabra (DCEXS), Barcelona, Spain) with PE-labeled goat anti-mouse though the NKL cell line (14) lacks KIR expression, it does ex- Ј F(ab )2 were used to characterize the NKL cell lines by flow cytometry. press two other class I-specific inhibitory receptors: CD94: ϩ KIR NKL cell lines were sorted for equivalent KIR expression with a NKG2A and LILRB1 (previously called LIR-1 and ILT2) (Fig. FACStar cell sorter and clone-Cyt software and hardware (BD Biosciences). 1A). To assess the potential contributions of these receptors to Cultured NK cells were derived by the coculture of PBMC with irradi- inhibit cytolysis, we measured the capacity of untransduced NKL ated RPMI 8866 cells as described (15). Following this expansion and cells to kill 721.221 cells (a class I-negative B cell line) and stable subsequent enrichment by the MACS NK Cell Isolation kit (Miltenyi Bio- ϩ Ϫ transfectants of 721.221 expressing selected class I allotypes. NKL tec) per the manufacturer’s instructions, 3DL1*002 /LILRB1 NK cells cells kill 721.221 cells. This lysis was partially inhibited by target were sorted with a FACStar cell sorter and maintained in RPMI 1640 medium supplemented with 10% FBS, 2 mM glutamine (Invitrogen Life cell expression of HLA-B*5101, but not HLA-A*0301 (Fig. 1B)at Technologies), 100 U/ml penicillin/streptomycin (Invitrogen Life Technol- a difference that is statistically significant ( p Ͻ 0.01). Although ogies), and 100 U/ml rIL-2 (National Institutes of Health, National Cancer HLA-A*0301 does have a signal sequence that is permissive for Institute Preclinical Repository, Frederick, MD). CD94A:NKG2A ligand expression (19) and has been shown to partially inhibit lysis through LILRB1 (20), we did not observe Cytolytic assays inhibition. Cell killing assays (i.e., 4-h 51Cr release assays) were performed as de- Inhibition by B*5101 was due to its binding to LILRB1, which scribed (16) using KIR transduced NKL cell lines or polyclonal NK cell was seen from its abrogation when either anti-LILRB1 mAb or Downloaded from 51 lines as effectors and Cr-labeled target cells: 721.221 (untransfected) and anti-class I-specific mAb was added to the cytolytic assay (Fig. 721.221 (transfectants) expressing individual HLA class I allotypes that have been previously described (7). PE-conjugated, pan-HLA-specific 1B). Anti-CD94:NKG2A mAb had no effect, either when added mAb W6/32 (17) was used to assess and sort for equivalent class I expres- alone or in combination with anti-LILRB1 Abs, showing this sion on the 221 transfectants. Unconjugated Abs (anti-HLA mAb DX17 receptor does not contribute to B*5101-mediated inhibition. (DNAX), anti-LILRB1 mAb HP-F1, and anti-KIR3DL1 mAb DX9) were This finding is consistent with earlier studies indicating that used as blocking reagents in cell killing assays at a concentration of 25 http://www.jimmunol.org/ ␮g/ml per well. B*5101 does not have a signal sequence permissive for CD94: NKG2A ligand expression (19). From these results we con- Data analysis clude, LILRB1 is the only inhibitory receptor on NKL cells that recognizes B*5101, and its effect can be blocked with specific Where appropriate, results were presented as specific lysis or the percent- mAbs. These results are consistent with previous characteriza- age of inhibition of lysis (IOL) relative to the lysis of 721.221 to normalize lytic activity between the cell lines and adjust for variation in the lysis of tion of the NKL cell line (20). the 721.221 cell line. The percentage of IOL was calculated with the following formula: [1 Ϫ (percent-specific lysis of target cells)/(mean of per- KIR3DL1 transduced NKL cell lines reproduce predicted HLA cent-specific lysis of 721.221)] ϫ 100. Additionally, some results were specificities presented as the relative percentage of IOL compared with lysis by a con- Retroviral transduction was used to make lines of NKL cells by guest on September 25, 2021 trol NKL cell line. All effectors lysed untransfected 221 target cells at levels exceeding 30% at an E:T ratio of 20:1, and the background spon- that stably express 3DL1*002 and 3DL1*007. Cell sorting was taneous release for all targets was Ͻ15%. used to produce transductants expressing comparable amounts A predicted model of 3DL1*002 (Accession no. U31416) was generated of 3DL1*002 and 3DL1*007 at the cell surface (Fig. 2A). Using with Swiss Model (͗www.expasy.org/swissmod/SWISS-MODEL.html͘) anti-KIR3DL1 Ab titration we observed no difference in Ab (18). In this model the D0 domain was modeled separately from the D1 and D2 domains and the complete structure was assembled and viewed staining between the two KIR3DL1 variants (data not shown), using Deep View and MolScript version 2.1 programs, respectively suggesting equivalent Ab affinities. KIR transduced and un- (͗www.avatar.se/molscript.html͘). transduced NKL cells were compared for their capacity to kill

FIGURE 1. Inhibition of NKL cells by B*5101 is mediated by LILRB1. A, Flow cytometric analysis shows that NKL cells express CD94, NKG2A, and LILRB1 at the cell surface but not KIR. Staining with receptor-speciﬁc mAb is given by the ﬁlled histogram and staining with an isotype control mAb by the open histogram. B, Lysis of 721.221 cells by NKL cells is inhibited by B*5101 in a LILRB1-dependent fashion. Shown is the lysis of three target cells: 721.221 cells (f) and 721.221 cells transfected with HLA-A*0301 (u) or HLA-B*5101 (Ⅺ) in the presence and absence of several mAbs. The E:T ratio was 20:1. Both anti-HLA class I and anti-LILRB1 mAbs abrogate the HLA-B*5101-mediated inhibition of cytolysis. No abrogation of lysis was caused by anti-CD94, although this mAb binds to NKL cells as shown in A. The assay shown represents two independent experiments in which SDs are indicated. 5224 POLYMORPHISM MODULATES INHIBITORY KIR3DL1 FUNCTION

721.221 cells transfected with B*5101, a Bw4ϩ ligand for the KIR3DL1 receptor, and with B*1502, a Bw6ϩ allotype. As expected, B*5101 elicited stronger lysis inhibition from NKL cells expressing the KIR3DL1 variants than from untransduced NKL cells (Fig. 2B). For NKL cells expressing either 3DL1*002 or 3DL1*007, stronger lysis inhibition occurred with B*5101 than with B*1502, although the latter was repro- ducible. Gumperz et al. (7) also found that some KIR3DL1ϩ NK cell clones were specifically inhibited by B*1502, but these same clones were significantly more inhibited by Bw4 allotypes (7). Thus, our data were consistent with previous descriptions of KIR3DL1 specificity for Bw4 HLA-B. Based on these data and previous descriptions of LILRB1 recognition of HLA-B allotypes (20, 21), we attributed inhibition of untransduced NKL cells to LILRB1 expression. However the observed differences in response between KIR3DL1 transduced and untransduced NKL cells were not due to differences in LILRB1 expression because LILRB1 expression was equivalent for

transduced and untransduced NKL cells (Fig. 2C). Downloaded from With this in vitro system, we replicated KIR3DL1 speciﬁc- ities previously described in the literature; but we had not established that KIRϩ cell lines responded like NK cells derived from peripheral blood. For this reason, we tested KIR3DL1ϩ peripheral blood NK cells with representatives from the same ϩ

panel of HLA transfectants as well as an additional Bw4 http://www.jimmunol.org/ HLA-B transfectant, B*2705. We derived polyclonal 3DL1*002ϩ, LILRB1Ϫ NK cells from the peripheral blood of a donor in whom 3DL1*002 was the only expressed 3DL1 receptor. Following in vitro expansion and sorting we verified the 3DL1*002ϩ, LILRB1Ϫ phenotype of the NK cells using flow cytometry (Fig. 2D). The response of these primary NK cells was consistent with the specificities of the 3DL1*002ϩ NKL cells (Fig. 2E). The ranking of the inhibitory response elicited relative to 721.221 by guest on September 25, 2021 lysis was B*5101ϾB*2705ϾB*1502 from greatest to least lysis inhibition consistent with data from previous studies (7, 22). None of these class I allotypes (i.e., B*5101, B*2705, or B*1502) had a canonical signal sequence that was permissive for CD94:NKG2A ligand expression (19). Thus, we found that FIGURE 2. KIR3DL1 transduced cells express equivalent levels of KIR some HLA-B ligands for 3DL1*002 elicited stronger inhibitory and are lysis inhibited by HLA-Bw4ϩ B*5101 similar to KIR3DL1ϩ pe- responses than others. ripheral blood NK cells. A, Expression of two KIR3DL1 allotypes on retrovirally transduced NKL cells in flow cytometry using the PE-conjugated Differences between 3DL1*002 and 3DL1*007 alter receptor KIR3DL1 specific mAb, DX9. B, KIR3DL1 allotypes, 3DL1*002 and strength 3DL1*007, reproduce predicted HLA specificities in a 4-h Cr release as- To distinguish inhibition due to KIR3DL1 from that due to say, which is representative of three independent experiments. Shown is LILRB1, assays were performed in the presence and absence of ࡗ the lysis of three target cells: 721.221 ( ), 721.221 cells transfected with specific mAbs. In the absence of receptor blocking Abs, lysis of all Bw6ϩ HLA-B*1502 (‚) or Bw4؉ HLA-B*5101 (f) in the absence of three NKL cell lines was inhibited, though less so for NKL than for anti-LILRB1 mAbs. All SDs are shown and where they are not apparent they are less than the size of the symbol. C, LILRB1 expression on the two transductants. In the presence of anti-LILRB1 mAb a clear Ͼ KIR3DL1 transduced and untransduced NKL cells is equivalent. Untrans- hierarchy of inhibition was seen: NKL expressing 3DL1*002 duced NKL (dotted line), NKL 3DL1*002 (dashed line), and NKL NKL expressing 3DL1*007 Ͼ untransduced NKL cells (Fig. 3). 3DL1*007 (solid line), and untransduced NKL stained with an isotype Thus, interaction of HLA-B*5101 with 3DL1*002 generates a control (filled histogram) are shown. D, Sorted and cultured NK cells iso- stronger inhibitory response than interaction of HLA-B*5101 with ϩ lated from the peripheral blood of one donor exhibit a 3DL1*002 / 3DL1*007. This difference was most evident by the percentage of Ϫ LILRB1 phenotype. Freshly isolated, unsorted NK cells (thick line), cul- lysis inhibition relative to lysis of 721.221 (Fig. 3B). Because the tured and sorted NK cells (filled histogram), and sorted NK cells stained ϩ Ϫ expression levels of the two KIR3DL1 allotypes were comparable, with an isotype control (dotted line) are shown. E, 3DL1*002 /LILRB1 this difference demonstrates that one or more of the amino acid NK cells are lysis inhibited by Bw4ϩ HLA-B allotypes similar to the substitutions that distinguish 3DL1*002 from 3DL1*007 make it a 3DL1*002 transduced NKL cells. In this donor the 3DL1*002 allotype was the only KIR3DL1 protein expressed at the cell surface. Shown is the lysis stronger receptor for HLA-B*5101. ࡗ ϩ of four target cells: 721.221 ( ), 721.221 cells transfected with Bw6 KIR3DL1 and LILRB1 both contribute to inhibit transduced (HLA-B*1502 (‚), Bw4ϩ HLA-B*2705 (Œ), or Bw4؉ HLA-B*5101 (f NKL cells in the absence of anti-LILRB1 mAbs. All SDs are shown and where they are not apparent they are less than the size of the symbol. These data are When anti-KIR3DL1 mAb was added to the cytolytic assay, lysis representative of two replicate experiments. inhibition caused by B*5101 binding to 3DL1*002 or 3DL1*007 The Journal of Immunology 5225

FIGURE 3. KIR3DL1 and LILRB1 both contribute to inhibit NK cell function. The killing of 721.221 cells and 721.221-B*5101 cells by NKL cells and NKL cells transfected with either 3DL1*002 or 3DL1*007 was compared in the absence and presence of speciﬁc mAbs in 4-h Cr release

assays at an E:T ratio of 20:1. The results are plotted as both a percentage Downloaded from specific lysis (A) and relative percentage of IOL (B) by 721.221-B*5101 targets in comparison to the lysis of untransfected 721.221 targets where the relative percentage of IOL is calculated using [1 Ϫ (percent-specific lysis of target cells)/(mean of percent-specific lysis of 721.221)] ϫ 100. The combination of anti-LILRB1 and anti-KIR3DL1 mAbs abrogated the inhibition to an extent greater than either Ab alone. Ab specific for CD56

served as a negative control, Ab speciﬁc for HLA class I as a positive http://www.jimmunol.org/ control. NKL (Ⅺ), NKL 3DL1*002 (f), and NKL 3DL1*007 (u) are shown. The data presented are the average of three separate experiments. ,ء p Ͻ 0.01 and ,ءء SEs are indicated. Statistical signiﬁcance shown where p Ͻ 0.05 are by t test.

FIGURE 4. 3DL1*002 is a stronger Bw4-specific inhibitory receptor than 3DL1*007. A, NKL cell lines expressing either 3DL1*002 or was partially blocked, whereas there was no affect on inhibition in 3DL1*007 were compared in their lysis of HLA B*5101 and HLA B*2705 the untransduced NKL parental cell line (Fig. 3). In contrast, when expressing 721.221 cells in 4-h Cr release assays at an E:T ratio of 20:1. In by guest on September 25, 2021 both anti-LILRB1 and anti-KIR3DL1 mAbs were included there this comparison, a control (untransduced NKL cells) was also included. was an equivalently strong blockade of inhibition of NKL cells The data are plotted as relative lysis inhibition compared with the lysis of expressing 3DL1*002 or 3DL1*007 and the parental cell line that class I-negative 721.221 cells. As done previously, anti-LILRB1 Ab was was comparable to that observed in the presence of anti-HLA class added to eliminate LILRB1-mediated lysis inhibition and anti-CD56 Ab I mAb. Thus, in the NKL cell transductants that express both was used as a control. The data presented are the average of three separate LILRB1 and KIR3DL1, lysis inhibition is the consequence of both experiments. NKL (Ⅺ), NKL 3DL1*002 (f), and NKL 3DL1*007 (u) are ,p Ͻ 0.01 is by t test. B ,ءء receptors recognizing the same HLA ligand. Individually, the in- shown. Statistical significance shown where hibition mediated by LILRB1 is stronger than that mediated by Class I expression on HLA transfectants using anti-HLA Ab, W6/32 PE- labeled. 721.221 (filled histogram), 721 B*5101 (solid line), and 721 KIR3DL1; however, when one of these receptors is blocked (e.g., B*2705 (dashed line). LILRB1) the other receptor (e.g., 3DL1*002) can compensate to inhibit lysis. Functional differences between the KIR3DL1 variants were only apparent when the effects of LILRB1 were removed by ing cell lines were more inhibited than untransduced NKL cells the presence of anti-LILRB1 Ab. When LILRB1 function was not in the presence of either a control Ab or no Ab ( p Ͻ 0.01) (Fig. blocked (i.e., in the presence of a control Ab or no Ab) we did not 4A). By comparison, B*2705 elicited no difference between observe differences between the KIR3DL1 variants. This outcome 3DL1*007-expressing and untransduced NKL cells, but did in- suggests that LILRB1 can compensate for functional differences crease the inhibition through 3DL1*002. The levels of class I between 3DL1*002 and 3DL1*007. expression were equivalent between the B*5101 and B*2705 transfectants (Fig. 4B). Thus, we attributed the differences in On interaction with HLA-Bw4, 3DL1*002 gives stronger inhibitory response to intrinsic differences in the HLA-B li- inhibition of cytotoxicity than 3DL1*007 gands. We found that B*5101 elicited a stronger inhibitory re- The removal of LILRB1-mediated inhibition revealed allotype sponse than B*2705. This finding confirms previous observa- differences in the inhibitory response to B*5101. Specifically, tions made by Cella et al. (22). cells expressing 3DL1*002 were more inhibited than those ex- In the presence of anti-LILRB1 Ab, we observed more marked pressing 3DL1*007. However, the question of whether this was differences among the KIR3DL1 variants in their inhibitory re- a general phenomenon or specific to 3DL1*002-HLA B*5101 sponse to B*5101 and B*2705. Both B*5101 and B*2705 elicited interactions remained unresolved. To address this issue, we significant inhibition through 3DL1*002 ( p Ͻ 0.01); however, compared the inhibitory responses generated by B*5101 to B*2705 did not elicit inhibition through 3DL1*007 (Fig. 4A). those generated by another Bw4ϩ HLA-B allotype, B*2705 Thus, 3DL1*007 either does not recognize or responds poorly to (Fig. 4). Again, we used anti-LILRB1 mAb to exclude the con- B*2705. Although 3DL1*007 was inhibited by B*5101, tributions of LILRB1 to lysis inhibition. As in previous exper- KIR3DL1*002 was more inhibited by this class I allotype. There- iments with B*5101, both 3DL1*002- and 3DL1*007-express- fore, 3DL1*002 was the stronger inhibitory receptor with more 5226 POLYMORPHISM MODULATES INHIBITORY KIR3DL1 FUNCTION than one Bw4ϩ HLA-B ligand, and this was most evident in the transmembrane domains, and 373 in the cytoplasmic tail (Fig. response to HLA-B*2705. 5A) (9). To determine the contribution of each position to the phenotypic difference we made a panel of six mutants, three The functional difference between 3DL1*002 and 3DL1*007 is from 3DL1*002 and three from 3DL1*007, in which each mu- determined by polymorphism at position 238 in the D2 domain tant had one position changed to the residue present in the other and position 320 in the transmembrane region KIR3DL1 allotype (Fig. 5B). These mutants were transduced The 3DL1*002 and 3DL1*007 allotypes differ at three positions into NKL cells and the transductants sorted to obtain cells hav- in the amino acid sequence: 238 in the D2 domain, 320 in the ing comparable levels of KIR3DL1 expression at the surface transmembrane region at the junction of the extracellular and (Fig. 5C). The mutant and the wild-type KIR3DL1 were then Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 5. Substitution at position 238 in the D2 domain and position 320 in the transmembrane domain make 3DL1*002 a stronger inhibitory receptor than 3DL1*007. A, Sequence alignments of the mature proteins reveal that 3DL1*002 and 3DL1*007 differ at three positions, one in each of the extracellular (D2), transmembrane (TM), and cytoplasmic (CYT) domains, respectively. The predicted boundary between extracellular and transmembrane domains is indicated by an arrowhead, where the extracellular region is to the left of the arrowhead and the transmembrane region is to the right. B, Six point mutants of 3DL1*007 and 3DL1*002 were made. Each had a residue swapped at one of the three positions, where the natural wild-type (WT) allotypes differ: 238 in the D2 domain (D2), 320 in the transmembrane region (TM), and 373 in the cytoplasmic tail (CYT). C, Expression of mutated KIR3DL1 allotypes is equivalent on transduced NKL cells: untransduced NKL (ﬁlled histogram), NKL 3DL1*002 WT (green line), NKL 3DL1*007 WT (pink line), NKL 3DL1*007 238R (orange line), NKL 3DL1*007 320I (purple line), NKL 3DL1*007 373E (navy blue line), NKL 3DL1*002 238G (black line), NKL 3DL1*002 320V (yellow line), and NKL 3DL1*002 373Q (light blue line). D, The capacity of NKL cells transfected with the point mutant KIR3DL1 to be inhibited by HLA-B*2705 (f) in the presence of anti-LILRB1 mAb was compared with untransfected NKL cells. Lysis of 721.221 cells (Ⅺ)inthe presence of anti-LILRB1 mAb was also included for comparison. E, In addition, the data from D were normalized to lysis inhibition of NKL cells where the relative percentage of IOL is calculated using [(percent lysis inhibition of effector Ϫ percent lysis inhibition of NKL)/(percent lysis inhibition of NKL)] ϫ 100. F, Three double mutants of 3DL1*002 were made, each of which had two of the three difference residues swapped from 3DL1*007. A triple mutant was also made, which converted 3DL1*002 to 3DL1*007. G, The capacity of NKL cells transfected with the double and triple mutant KIR3DL1 to be inhibited by HLA-B*2705 was compared with untransfected NKL cells. H, The data from G were also normalized to lysis inhibition of NKL3DL1*007 where the relative percentage of IOL was calculated by [(percent lysis inhibition of effector Ϫ percent lysis inhibition of NKL3DL1*007)/(percent lysis inhibition of NKL3DL1*007)] ϫ 100. All cytolytic assays were performed at an E:T ratio of 20:1 in the presence of anti-LILRB1 mAb. Data presented are the average of ﬁve (D and E) and three (G and H) separate 4-h Cr release assay experiments with SEs as indicated. The Journal of Immunology 5227 compared for their inhibitory capacity in cytolytic assays using 721.221-B*2705 target cells and inclusion of anti-LILRB1 mAb. The lysis obtained with transductants expressing KIR3DL1 variants and mutants was compared with that obtained with untransduced NKL cells. Target cells expressing HLA-B*2705 were selected for this assay based on their ability to differentially inhibit the wild-type KIR3DL1 variants relative to untransduced cells. HLA-B*2705 is in general a weaker inhibitor of KIR3DL1 than B*5101, and 3DL1*007 is a weaker receptor than 3DL1*002. Whereas 3DL1*002 engages B*2705 to inhibit lysis, the combination of HLA-B*2705 and 3DL1*007 gave no inhibition (Figs. 4A and 5, D and E). For a detailed comparison of the effects of the mutant and wild-type KIR3DL1 allotypes, the data were normalized to untransduced NKL cells (Fig. 5E). Mutants of 3DL1*007, with substitution at either position 238 or 320, engage B*2705 to give some inhibition. Conversely the reciprocal mutations in 3DL1*002 reduce its inhibitory capacity. Mutation at position 373 in either FIGURE 6. Residues 238 and 320 that give functional difference to 3DL1*002 or 3DL1*007 had no functional effect. Downloaded from 3DL1*002 and 3DL1*007 are not within the predicted ligand binding site. As none of the mutants having a single residue change altered A model of KIR3DL1 based upon the known structures for KIR2D is the phenotype completely, four further mutants of 3DL1*002 were shown. The ligand binding site for HLA-Bw4 is predicted to be formed by made: three had the various combination of two amino acid sub- interaction of the D1 and D2 domains as indicated by the arrow. Residue stitutions, the fourth had all three substitutions and served as a 238 is situated in a ␤-strand loop of the D2 domain but is distant from the control (Fig. 5F). As done previously, the mutants were sorted for ligand binding site; it is shown (boxed) as the arginine present in

equivalent KIR3DL1 expression (data not shown). To determine 3DL1*002. Residue 320 is in the transmembrane domain, which is not http://www.jimmunol.org/ the relative impact of these double and triple substitutions in shown in the schematic. 3DL1*002 the data were normalized to wild-type 3DL1*007 (Fig. 5, G and H). As predicted from the results obtained from the single A68 and HLA-B48 abrogates this binding (25–27). Apparent in residue mutants, the double mutant of 3DL1*002 with substitution the CD8-HLA co-crystal this polymorphism distorts a protrud- at positions 238 and 320 gave no inhibition, like 3DL1*007 and ing loop of the ␣3 domain, although the polymorphic residue at the triple mutant, whereas the two other double mutants retained position 245 is not directly involved in contact with CD8 (26). some inhibitory capacity. In conclusion, substitution at position Thus we speculate that similar conformational changes may ex- 238 in the D2 domain and position 320 in the transmembrane plain the impact of the KIR3DL1 polymorphism examined in region contribute to 3DL1*002 being a stronger inhibitory receptor this study. by guest on September 25, 2021 than 3DL1*007, with each position making a comparable contri- Although residue 238 is distant from the putative binding bution to the difference. site: in a loop of the D2 domain opposite the hinge between the To further understand how these residues influenced inhibitory D1 and D2 domains, a significant change at this position may function we generated a predicted molecular model of 3DL1*002 alter the conformation of the ligand binding loops. The substi- based on established KIR2D crystal structures (23, 24). In the tution of arginine for glycine at position 238 is a radical one: the KIR2D/HLA co-crystal structures ligand binding is localized to arginine being found only in 3DL1*002 (9). In comparison, the KIR residues in the interdomain hinge region. In our model we substitution of isoleucine in 3DL1*002 for valine in 3DL1*007 found that residue 238 in the D2 domain was distant from the at position 320 is a conservative one, but this position occurs at putative ligand-binding site in a loop opposite the interdomain the boundary between extracellular and transmembrane do- hinge region (Fig. 6). Thus, we propose that the enhanced inhib- mains. Thus, even conservative substitutions in this model itory effects of these residues are due to indirect influences on could significantly alter conformation of the extracellular re- ligand recognition, rather than direct ligand interactions. gion. In this manner, both residues 238 and 320 have the potential to indirectly alter conformation of the binding site and Discussion thereby influence inhibitory function. In this study we demonstrate that KIR polymorphism can serve to Conformational changes are not the only potential mechanism to alter the inhibitory response to a MHC class I ligand. When two explain the effects of KIR3DL1 polymorphism. An alternative allotypes of KIR3DL1 were expressed at similar levels on the sur- mechanism for contribution of positions 238 and 320 to KIR3DL1 face of NKL cells and challenged with the same HLA-Bw4 ligand, function is the facilitation of receptor oligomerization. Whereas 3DL1*002 was seen to be a stronger inhibitor than 3DL1*007. The researchers have speculated that KIR oligomerization occurs at the two allotypes differ by three amino acid substitutions, of which immune synapse, this has not been formally proven. Although sev- residue 238 in the D2 domain and residue 320 in the transmem- eral crystallographic structures have been determined for com- brane region contribute to the phenotypic difference in roughly plexes of HLA-C with KIR2D (23, 24), no structure for a equal part. The mechanism that underlies the altered response to KIR3DL1 has been solved, either alone or in complex with HLA- ligand is uncertain but seems more likely to have an indirect effect Bw4. Several lines of evidence, including the importance of resi- on ligand binding than a direct effect. due 80 in both HLA-B and HLA-C for KIR interaction (28, 29), A proposed mechanism that invokes indirect effects on ligand suggest that the D1 and D2 domains of KIR3DL1 bind to HLA- binding has precedence in HLA polymorphism that influences Bw4 in analogous manner to the KIR2D-HLA-C interaction. The CD8 binding. During T cell target engagement CD8 binds to the D0 domain of KIR3DL1 enhances the interaction in a manner that membrane proximal ␣3 domain of class I on the opposing cell. is poorly understood (30), but may also involve oligomerization of A naturally occurring single amino acid polymorphism in HLA- KIR-HLA-Bw4 complexes. 5228 POLYMORPHISM MODULATES INHIBITORY KIR3DL1 FUNCTION

In the co-crystal structure of KIR2DL2 complexed with HLA- viral vectors and Lewis Lanier for valuable discussions. We also thank the Cw3, two KIR2DL2 proteins (i.e., KIR-A and KIR-B) contact Stanford University FACS facility for sorting cells. each other through D2 (KIR-A) and D1 (KIR-B) domain interactions, respectively (23). Although amino acid residues in the Disclosures ␤-strand loops of the D2 domain (i.e., I200, L119, E122, and The authors have no financial conflict of interest. V118) of KIR-A participate in KIR oligomerization, these residues do not correspond directly with position 238 of KIR3DL1 in our References model of KIR3DL1 (Fig. 6). However, the presence of an addi- 1. Bauer, S., V. Groh, J. Wu, A. Steinle, J. H. Phillips, L. L. Lanier, and T. Spies. 1999. Activation of NK cells and T cells by NKG2D, a receptor for stress- tional extracellular domain (i.e., D0) may alter the conformation of inducible MICA. Science 285: 727–729. the D2 domain of KIR3DL1. Thus, receptor oligomerization in- 2. Valiante, N. M., M. Uhrberg, H. G. Shilling, K. Lienert-Weidenbach, volving position 238 cannot be excluded completely without fur- K. L. Arnett, A. D’Andrea, J. H. Phillips, L. L. Lanier, and P. Parham. 1997. Functionally and structurally distinct NK cell receptor repertoires in the periph- ther evaluation. eral blood of two human donors. Immunity 7: 739–751. In addition, the idea that amino acid residues in the transmem- 3. Lanier, L. L. 1998. NK cell receptors. Annu. Rev. Immunol. 16: 359–393. brane domain facilitate homotypic interactions during receptor oli- 4. Wilson, M. J., M. Torkar, A. Haude, S. Milne, T. Jones, D. Sheer, S. Beck, and J. Trowsdale. 2000. Plasticity in the organization and sequences of human KIR/ gomerization has precedence in other biological systems. In the ILT gene families. Proc. Natl. Acad. Sci. USA 97: 4778–4783. gp41 envelope protein of HIV-1 a single isoleucine residue in the 5. Uhrberg, M., N. M. Valiante, B. P. Shum, H. G. Shilling, K. Lienert-Weidenbach, transmembrane domain is essential for receptor oligomerization B. Corliss, D. Tyan, L. L. Lanier, and P. Parham. 1997. Human diversity in killer cell inhibitory receptor genes. Immunity 7: 753–763. and biological function and without it oligomerization does not 6. Colonna, M., G. Borsellino, M. Falco, G. B. Ferrara, and J. L. Strominger. 1993. occur (31). It is reasonable to predict that residue 320 of KIR3DL1 HLA-C is the inhibitory ligand that determines dominant resistance to lysis by Downloaded from NK1- and NK2-specific natural killer cells. Proc. Natl. Acad. Sci. USA 90: may play a similar role. 12000–12004. In summary, we propose two possible mechanisms by which 7. Gumperz, J. E., V. Litwin, J. H. Phillips, L. L. Lanier, and P. Parham. 1995. The naturally occurring polymorphism at positions 238 and 320 may Bw4 public epitope of HLA-B molecules confers reactivity with natural killer cell clones that express NKB1, a putative HLA receptor. J. Exp. Med. 181: influence KIR3DL1 function. In one proposed mechanism these 1133–1144. residues determine distantly the conformation of ligand binding 8. Pende, D., R. Biassoni, C. Cantoni, S. Verdiani, M. Falco, C. di Donato, L. Accame, C. Bottino, A. Moretta, and L. Moretta. 1996. The natural killer cell loops, and in another they determine receptor on receptor interac- http://www.jimmunol.org/ receptor specific for HLA-A allotypes: a novel member of the p58/p70 family of tions at the cell surface. Based on our data, either of these models inhibitory receptors that is characterized by three immunoglobulin-like domains provides a reasonable framework for understanding KIR3DL1 and is expressed as a 140-kD disulphide-linked dimer. J. Exp. Med. 184: 505–518. polymorphism in the context of inhibitory function. 9. Gardiner, C. M., L. A. Guethlein, H. G. Shilling, M. Pando, W. H. Carr, To eliminate the confounding effects of differing levels of R. Rajalingam, C. Vilches, and P. Parham. 2001. Different NK cell surface phe- cell surface expression we deliberately selected and studied notypes defined by the DX9 antibody are due to KIR3DL1 gene polymorphism. J. Immunol. 166: 2992–3001. NKL transductants that expressed equivalent levels of 10. Pando, M. J., C. M. Gardiner, M. Gleimer, K. L. McQueen, and P. Parham. 2003. 3DL1*002 and 3DL1*007. This showed that 3DL1*007 is an The protein made from a common allele of KIR3DL1 (3DL1*004) is poorly inherently weaker Bw4 receptor than 3DL1*002. In nature expressed at cell surfaces due to substitution at positions 86 in Ig domain 0 and 182 in Ig domain 1. J. Immunol. 171: 6640–6649. by guest on September 25, 2021 3DL1*007 is expressed at lower levels on human peripheral 11. Norman, P. 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HLA-E-bound peptides influence recognition by inhibi- chinery amplifies the inhibitory signal. In another model, either tory and triggering CD94/NKG2 receptors: preferential response to an HLA-G- derived nonamer. Eur. J. Immunol. 28: 2854–2863. LILRB1 or KIR3DL1 functions as an adhesion molecule (analo- 20. Colonna, M., F. Navarro, T. Bellon, M. Llano, P. Garcia, J. Samaridis, gous to CD8) and prolongs the ligand binding of the receptor that L. Angman, M. Cella, and M. Lo´pez-Botet. 1997. A common inhibitory receptor initiates signaling. Distinguishing these models will require further for major histocompatibility complex class I molecules on human lymphoid and myelomonocytic cells. J. Exp. Med. 186: 1809–1818. analysis to compare lysis inhibition with receptors mutated to 21. Cosman, D., N. Fanger, L. Borges, M. Kubin, W. Chin, L. Peterson, and eliminate signaling function specifically. M. L. Hsu. 1997. 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